Compositions and methods for treating post-operative complications of cardiopulmonary surgery

ABSTRACT

Disclosed herein are compositions and methods for treating damage inflicted by use of a cardio-pulmonary bypass (CPB) machine, particularly excessive bleeding and multi organ failure, by administering a pharmaceutical composition comprising alpha-1 antitrypsin (AAT).

CROSS REFERENCE TO RELATED APPLICATIONS

Benefit is claimed to U.S. Provisional Patent Application No.61/823,655, filed May 15, 2013; the contents of which are incorporatedby reference in their entirety herein.

FIELD

Disclosed herein are compositions and methods for treating damageinflicted by use of a cardio-pulmonary bypass (CPB) machine,particularly excessive bleeding and multi organ failure, byadministering a pharmaceutical composition comprising alpha-1antitrypsin (AAT).

BACKGROUND

Open heart surgery using cardiopulmonary bypass (CPB) is one of the mostcommon surgical procedures performed today. Approximately 1,000,000operations are conducted worldwide each year, of which 500,000 areconducted in the United States alone. Use of CPB can profoundly alterhaemostasis as well as injure vital organs, predisposing patients tomajor haemorrhagic complications and multi organ failure.

Excessive post-operative bleeding necessitating additional surgeryoccurs in 7% of patients undergoing CPB. Re-operation for bleedingincreases hospital mortality, substantially increases post-operativehospital stay and has a sizeable effect on health care costs.

Concerns about transfusion safety, blood product shortages andincreasing blood hank costs have generated increased interest inadopting risk-limiting strategies for post-operative bleeding. In orderto prevent damage from CBP, several anti-inflammatory agents have beenemployed during cardiac surgery; and most commonly Aprotinin.

Despite its ability to reduce postoperative bleeding, the use ofAprotinin was abandoned worldwide in 2007 after studies suggesting thatits use increased the risk of complications or death. Aprotinin waswithdrawn from distribution because studies demonstrated a higherincidence of myocardial infarction in comparison to those treated usingother available agents (such as hexacapron). Since the withdrawal ofAprotinin from use, high risk cardiac procedures are prone to high rateof post-operative bleeding. There is thus a continuing need to developtreatments which can be used in conjunction with CPB and effectivelyreduce post-operative blood loss.

SUMMARY

Provided herein are compositions comprising a therapeutically effectiveamount of alpha-1 antitrypsin (AAT-1), or a functional variant thereof,for use in preventing or treating injury to a subject during orresultant from cardiac surgery, and particularly cardiac surgery usingcardiopulmonary bypass.

Additionally described herein are methods for treating or preventinginjury during or resultant from cardiac surgery in a subject, such ascardiac surgery using cardiopulmonary bypass by administering to thesubject a composition comprising a therapeutically effective amount ofalpha-1 antitrypsin (AAT-1), or a functional variant thereof.

The foregoing and other objects, features, and advantages will becomemore apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic and/or amino acid sequences provided herewith are shownusing standard letter abbreviations for nucleotide bases, and threeletter code for amino acids, as defined in 37 C.F.R. 1.822. Only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. The Sequence Listing is submitted as an ASCII text file namedMORR 4 2 Seq list_ST25.txt, created May 12, 2014, about 8 KB, which isincorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO. 1 is the cDNA sequence of human alpha-1 antitrypsintranscript.

SEQ ID NO. 2 is the amino acid sequence of human alpha-1 antitrypsinprotein.

DETAILED DESCRIPTION I.

Abbreviations AAT-1 Alpha-1 Anti Trypsin ACT Activated Clotting Time AKIAcute Kidney Injury BAL Broncho alveolar lavage CABG Coronary ArteryBypass Grafting CPB Cardio Pulmonary Bypass CVP Central Venous PressureHBV Hepatitis B Virus HCV Hepatitis C Virus KIM Kidney injury moleculeMI Myocardial Infarction N-GAL Neutrophil gelatinase

II. Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Although methodsand materials similar or equivalent to those described herein can beused in the practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”The abbreviation, “e.g.” is derived from the Latin exempli gratia, andis used herein to indicate a non-limiting example. Thus, theabbreviation “e.g.” is synonymous with the term “for example.”

In case of conflict, the present specification, including explanationsof terms, will control. In addition, all the materials, methods, andexamples are illustrative and not intended to be limiting.

Abnormal: Deviation from normal characteristics. Normal characteristicscan be found in a control, a standard for a population, etc. Forinstance, where the abnormal condition is an injury or physicalresponse, such as injury resultant from cardiac surgery employingcardiopulmonary bypass, a few appropriate sources of normalcharacteristics might include an individual or a population standard ofa collection of individuals who are not suffering from the injury orexperiencing the particular physical response. Controls or standardsappropriate for comparison to a sample, for the determination ofabnormality, include samples believed to be normal as well as laboratorydetermined values, even though such values are possibly arbitrarily set,and keeping in mind that such values may vary from laboratory tolaboratory. Laboratory standards and values may be set based on a knownor determined population value and may he supplied in the format of agraph or table that permits easy comparison of measured, experimentallydetermined values.

Administration: The introduction of a composition into a subject by achosen route. Administration of an active compound or composition can beby any route known to one of skill in the art. Administration can belocal or systemic. Local administration includes routes ofadministration typically used for systemic administration, for exampleby directing intravascular administration to the arterial supply for aparticular organ. Thus, in particular embodiments, local administrationincludes intra-arterial administration and intravenous administrationwhen such administration is targeted to the vasculature supplying aparticular organ. Local administration also includes the incorporationof active compounds and agents into implantable devices or constructs,such as vascular stents or other reservoirs, which release the activeagents and compounds over extended time intervals for sustainedtreatment effects.

Systemic administration includes any route of administration designed todistribute an active compound or composition widely throughout the bodyvia the circulatory system. Thus, systemic administration includes, butis not limited to intra-arterial and intravenous administration.Systemic administration also includes, but is not limited to, topicaladministration, subcutaneous administration, intramuscularadministration, or administration by inhalation, when suchadministration is directed at absorption and distribution throughout thebody by the circulatory system.

Cardiac surgery: Any surgical procedure involving treatment of thecardiovascular system of a subject, and which can impair or temporarilystop normal cardiovascular function. In particular examples, cardiacsurgery requires the heart of the subject to be stopped, but this is notan absolute requirement of all forms of cardiac surgery. Particularexamples of cardiac surgery include coronary artery bypass graftingsurgery, aortic valve replacement or repair, mitral valve replacement orrepair, tricuspid valve replacement or repair, ascending aortareplacement, heart transplantation, lung transplantation or anycombination of the above.

Cardiopulmonary bypass (CPB): surgical technique for maintaining bloodcirculation and oxygenation when heart function is impaired ortemporarily stopped. CPB is achieved through use of a pump. Inparticular examples, the pump is known as a “heart-lung machine,” CPBpump, or CPB machine. In other examples, a type CPB is also known as“extracorporeal membrane perfusion.”

Functional fragments and variants of a polypeptide: Included are thosefragments and variants that maintain one or more functions of the parentpolypeptide, such as a functional fragment or variant of AAT-1. It isrecognized that the gene or cDNA encoding a polypeptide can beconsiderably mutated without materially altering one or more thepolypeptide's functions. First, the genetic code is well-known to bedegenerate, and thus different codons encode the same amino acids.Second, even where an amino acid substitution is introduced, themutation can be conservative and have no material impact on theessential functions of a protein. Third, part of a polypeptide chain canbe deleted without impairing or eliminating all of its functions.Fourth, insertions or additions can be made in the polypeptide chain forexample, adding epitope tags, without impairing or eliminating itsfunctions. Other modifications that can be made without materiallyimpairing one or more functions of a polypeptide include, for example,in vivo or in vitro chemical and biochemical modifications or theincorporation of unusual amino acids. Such modifications include, forexample, acetylation, carboxylation, phosphorylation, glycosylation,ubiquination, labeling, e.g., with radionucleides, and various enzymaticmodifications.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Variant amino acid sequences may, for example, be 80%, 85%, 90% or even95% or 98% identical to the native AAT-1 amino acid sequence. Programsand algorithms for determining percentage identity can be found at theNCBI website.

Injectable composition: A pharmaceutically acceptable fluid compositioncomprising at least one active ingredient, for example, a protein,peptide, or antibody. The active ingredient is usually dissolved orsuspended in a physiologically acceptable carrier, and the compositioncan additionally comprise minor amounts of one or more non-toxicauxiliary substances, such as emulsifying agents, preservatives, pHbuffering agents and the like. Such injectable compositions that areuseful for use with the compositions of this disclosure areconventional; appropriate formulations are well known in the art.

Organ injury: Impairment of normal organ function in a mammaliansubject, including human and veterinary subjects. Organ injury asunderstood herein does not require complete loss of organ function. Inparticular examples, loss of specific organ function is diagnosed bydetection of biological markers. For example, detection of liver enzymesis one indicator of liver injury.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton.Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds herein disclosed.In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Preventing or treating an injury: Preventing an injury refers toinhibiting the full development of an injury or pathological condition,for example inhibiting excessive post-operative bleeding or organ injuryin a person who has or is undergoing cardiac surgery. Treatment refersto a therapeutic intervention that ameliorates a sign or symptom of theinjury or pathological condition after it has begun to develop.

Resultant: An effect of a causative event is said to be “resultant” fromthat event. For example, in particular subjects, excessive bleeding isresultant from use of cardiopulmonary bypass in cardiac surgery. Inparticular examples, a resultant effect may immediately follow thecausative event. In other examples, a resultant effect develops as adelayed response to the event. For example, certain organ damage may beresultant from use of cardiopulmonary bypass on a subject during cardiacsurgery, but the extent of the damage may not be fully apparent forhours or even days after the surgery.

Subject: Living multi-cellular organisms, including vertebrateorganisms, a category that includes both human and non-human mammals.

Therapeutically effective amount: A quantity of compound sufficient toachieve a desired effect in a subject being treated. An effective amountof a compound may be administered in a single dose, or in several doses,for example daily, during a course of treatment. However, the effectiveamount will be dependent on the compound applied, the subject beingtreated, the severity and type of the affliction, and the manner ofadministration of the compound.

III. Overview of Several Embodiments

Described herein are compositions including a therapeutically effectiveamount of alpha-1 antitrypsin (AAT-1), or a functional variant thereof,for use in preventing or treating injury to a subject during orresultant from cardiac surgery, and particularly from the use ofcardiopulmonary bypass. In particular embodiments, the injury isexcessive post-operative bleeding or organ injury.

In particular embodiments, the composition can be administered to thesubject before the cardiac surgery, during the cardiac surgery, afterthe cardiac surgery or a combination thereof, in those embodimentswherein the composition is administered to the subject in multipledoses.

In some embodiments of the described composition, the concentration ofAAT-1, or the functional variant thereof, is 1 gram in 50 cc sterilefluid in the form of a sterile or physiologically isotonic aqueoussolution.

In some embodiments of the described composition, the AAT-1 isadministered to the subject at a concentration of 30 to 300 mg per kgbody weight per day. In other embodiments of the described composition,the AAT-1 is administered to the subject at a concentration of 60 to 100mg per kg body weight per day.

In particular embodiments, the composition is administered as a singledose. In other embodiments, the composition is administered in multipledoses.

In some embodiments, the sole active ingredient of the composition isAAT-1. In other embodiments, the composition includes multiple activeingredients, and particularly at least one additional active ingredientfor treatment of injury resultant from cardiac surgery. In still otherembodiments, the additional one or more active ingredients isadministered to the subject in an additional composition, which can beadministered to the subject prior to, concurrent with, or afteradministration of the composition comprising AAT-1.

In particular embodiments the subject is human. In other embodiments,the subject is a veterinary subject.

Also described herein are methods for treating or preventing injuryduring or resultant from cardiac surgery in a human or veterinarysubject, by administering to the subject a composition comprising atherapeutically effective amount AAT-1, or a functional variant thereof.

Particular embodiments of the described methods are directed totreatment or prevention of the injury resulting from use of cardiacbypass, including excessive post-operative bleeding or organ injury.

In particular embodiments, the composition is administered to thesubject before the cardiac surgery, during the cardiac surgery, afterthe cardiac surgery or a combination thereof.

In some embodiments, the concentration of AAT-1, or functional variantthereof, in the composition is 1 gram in 50 cc sterile fluid in the formof a sterile or physiologically isotonic aqueous solution.

In particular embodiments, the AAT-1, or functional variant thereof, isadministered to the subject at a concentration of 30 to 300 mg per kgbody weight per day. In other embodiments, the AAT-1 is administered tothe subject at a concentration of 60 to 100 mg per kg body weight perday.

In some embodiments, the AAT-1-containing composition is administered asa single dose. In other embodiments, the AAT-1-containing composition isadministered in multiple doses.

In further embodiments, of the described methods, the compositioncomprises at least one additional composition for treatment of injuryresultant from cardiac surgery. In other embodiments, the methodscomprise administration of at least one additional composition whichcontain one or more active ingredients for treatment of injury resultantfrom cardiac surgery, which is administered to the subject prior to,concurrent with, or after administration of the composition comprisingAAT-1.

IV. Cardiopulmonary Bypass

Cardiopulmonary bypass (CPB) during cardiac surgery elicits generalizednon-specific systemic inflammatory response syndrome (SIRS), whichinitiates the activation of cytokine, complement, andcoagulation-fibrinolytic cascades. In approximately 1% of all patients,depending on the number of organs involved, SIRS may result in severemulti-organ failure (MOF), having a mortality rate of 40-98%. Strategiesused to attenuate the effects of SIRS focus on optimization ofanaesthesiological, surgical, and CPB techniques.

During CPB, passage of blood through plastic tubing and through anoxygenator activates the clotting cascade, including activation ofcomplement, cytokines, platelets, neutrophils, adhesion molecules, mastcells, and multiple inflammatory mediators. This can generatemulti-organ system dysfunction that can manifest in a subject aspost-operative respiratory failure, myocardial dysfunction, renalinsufficiency, and neurocognitive defects.

The mechanism of damage to the lungs during cardiopulmonary bypass isunique. During CBP, blood flow through pulmonary circulation is minimal.This is followed by sequestration of neutrophils in the pulmonarycapillary bed probably secondary to the lack of blood flow and theactivation of pro inflammatory cytokines. The sequestered neutrophils,through the release of proteolytic enzymes, cause endothelial cellswelling, plasma and protein extravasation into the interstitial tissue,congestion of the alveoli with plasma, erythrocytes and inflammatorydebris. Coagulation and inflammation are closely linked duringcardiopulmonary bypass through networks of both humoral and cellularcomponents including activation of proteases of the clotting andfibrinolytic cascades.

V. Methods for Inhibiting or Preventing Injury During or Resultant FromCardiac Surgery

Described herein are methods of treating or preventing injury to asubject associated with cardiac surgery. The described methods involveadministration of a composition comprising an effective amount of atleast one dose of alpha 1 anti trypsin (AAT-1) prior to, concurrentwith, or following cardiac surgery.

In particular embodiments the AAT-1-containing composition can beadministered to the subject prior to the start of cardiac surgery suchas 1, 2, 3, 4, 5 or more hours before surgery, including prior toadministration of anesthesia or during pre-operative preparations. Inother embodiments, the AAT-1-containing composition can be administeredduring cardiac surgery. In still other embodiments, the AAT-1 containingcomposition can be administered following the surgery, such as 1, 2, 3,4, 5 or more hours after surgery, or 1, 2, 3, 4, 5 or more daysfollowing surgery

In particular embodiments, the cardiac surgery involves use ofcardiopulmonary bypass. In such embodiments, the at least one dose ofAAT-1 is administered to the subject prior, current with, or after useof a cardiopulmonary bypass machine, but while the surgery is ongoing.

Cardiac surgery employing CPB can result in particular physiologicalpathologies in a subject, such as excessive post-operative bleedingand/or organ damage. The compositions and methods described herein cantreat such pathologies, and therefore decrease the severity of thepost-operative bleeding and/or organ damage. In particular examples,administration of an AAT-1 containing composition to a subject prior to,during, or following cardiac surgery employing CPB can prevent theinjury. In such examples, post-operative bleeding is prevented fromoccurring as is organ damage. The development of post-operative bleedingand organ damage is determined by standard methods known to the art.

Alpha-1 Antitrypsin (AAT-1)

AAT-1 is a plasma-derived protein belonging to the family of serineproteinase inhibitors. AAT-1 is synthesized primarily in the liver, andto a lesser extent in other cells, including macrophages, intestinalepithelial cells and intestinal Paneth cells. In the liver, AAT-1 isinitially synthesized as a 52 kD precursor protein that subsequentlyundergoes post translational glycosylation at three asparagine residues,as well as tyrosine sulfonation. The resulting mature protein issecreted as a 55 kD native single-chain glycoprotein. AAT-1, is alsoknown as SERPINA-1. Nucleotide and amino acid sequences of human AAT-1are available on-line at ncbi.nlm.nih.gov/nuccore/189163524 andncbi.nlm.nih.gov/protein/NP_000286, respectively, and are includedherein as SEQ ID NOs: 1 and 2.

AAT-1 is associated with control of tissue destruction by endogenousserine proteinases, and is the most prevalent serine proteinaseinhibitor in blood plasma. AAT-1 inhibits, inter alia, trypsin,chymotrypsin, various types of elastases, skin collagenase, renin,urokinase and proteases of polymorphonuclear lymphocytes.

Patients with low circulating levels of AAT-1 are at increased risk forlung, liver, and pancreatic diseases, particularly emphysema.Accumulating data suggests that besides its ability to inhibit serineproteases, AAT-1 possesses independent anti-inflammatory andtissue-protective effects. AAT-1 modifies dendritic cell maturation andpromotes regulatory T-cell differentiation, induces interleukin (IL)-1receptor antagonist and IL-10 release, protects various cell types fromcell death, inhibits caspases-1 and -3 activity and inhibits IL-1production and activity.

Importantly, and contradictory to classic immune-suppressants. AAT-1allows undeterred isolated T-lymphocyte responses. AAT-1 is currentlyused therapeutically for the treatment of pulmonary emphysema inAAT-1-deficient patients. AAT-1 deficiency is a genetic condition thatincreases the risk of developing a variety of diseases includingpulmonary emphysema (Laurell and Eriksson Scand J Clin lab Inves 1963.151132-140). AAT-1 deficiency is a result of mutations in theAAT-1-encoding gene (proteinase inhibitor (Pi) gene). Purified AAT-1 hasbeen approved for replacement therapy (also known as “augmentationtherapy”) in such patients deficient in endogenous AAT-1.

AAT-1 is currently administered intravenously, andcommercially-available AAT-1 preparations can be used in the methods andcompositions described herein. For example, AAT-1 marketed undertradenames including Aralast® (Baxter Healthcare Corporation, WestlakeVillage, Calif.); Zemaira® (CSL Behring, King of Prussia, Pa.);Prolastin® (Grifols Therapeutics Inc., Clayton, N.C.); Trypsone®(Evaluate, Ltd); and Alfalastin®, and which are human AAT-1 formulationsindicated for augmentation therapy in patients congenital deficiency ofAAT-1 with clinically evident emphysema, can be used as describedherein.

In addition to the commercially-available preparations, compositionscomprising AAT-1 can be produced by standard protein expression andpurification methodology known to the art and formulated foradministration as described herein. It is also appreciated thatfunctional variants of AAT-1 can be produced by standard methods ofmutagenesis, which will maintain the activity of the wild type protein,and can be used in the compositions and methods described herein. Suchfunctional variants can be identical in sequence to wild type AAT-1 byat least 98%, at least 95%, at least 90%, at least 85%, at least 80%, oreven less than 80% identical.

Combination Therapies

In particular embodiments of the compositions and methods describedherein. AAT-1 is combined with at least one additional active agent totreat or prevent injury resultant from excessive post-operative bleedingand/or organ damage. Non-limiting examples of active compounds fordecreasing post-operative bleeding include fresh frozen plasma,platelets, cryoprecipitate, and alpha aminocaproic acid. Non-limitingexamples of active compounds and procedures that can be used to decreaseorgan damage include steroids, and leucocyte depletion methods.

In some embodiments, the combination of AAT-1 and at least oneadditional active agent is administered to a subject in a singlecomposition. In particular examples, the combination compositions areformulated so that the component active ingredients are simultaneouslyavailable in the subject in an active form. In other examples, thecomponent active ingredients are formulated such that the components aresequentially available in an active form to the subject. For example,although administered simultaneously, the AAT-1 might produce thedesired effect prior to the at least one additional compound.

In other embodiments, combinations of AAT-1 and at least one additionalactive agent can be administered to a subject in multiple compositions,one containing AAT-1 and at least one additional composition containingthe at least one additional active agent. The timing and order ofadministration of such multiple compositions can vary, such as prior to,during, and after cardiac surgery, as described herein. In particularexamples, the AAT-1-containing composition is administered prior to theadditional composition. In other examples, the AAT-1-containingcomposition is administered simultaneously with the additionalcomposition. In still other embodiments, the AAT-1-containingcomposition is administered after the additional composition. It iscontemplated that when administered at separate times, significant timemay elapse between administration of the at least two compositions, suchas several hours, several days or even longer.

Pharmaceutical Compositions and Modes of Administration

The AAT-1 and other active agents for use in the described compositionsand methods can be supplied in any pharmaceutically acceptablecompositions. As described herein, AAT-1 is currently commerciallyavailable in several intravenous formulations.

Additionally, the pharmaceutical compositions specifically contemplatedin the present disclosure can include pharmaceutically acceptable acidor base addition salts. The phrase “pharmaceutically acceptable acid orbase addition salts” includes therapeutically active non-toxic acid andnon-toxic base addition salt forms which at least some of the activeagents described herein can form. Such compounds which have basicproperties can be converted in their pharmaceutically acceptable acidaddition salts by treating said base form with an appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric;nitric; phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic,malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic,tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic,p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and thelike acids.

Those active agents which have acidic properties may be converted intheir pharmaceutically acceptable base addition salts by treating saidacid form with a suitable organic or inorganic base. Appropriate basesalt forms comprise, for example, the ammonium salts, the alkali andearth alkaline metal salts, e.g. the lithium, sodium, potassium,magnesium, calcium salts and the like, salts with organic bases, e.g.the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.

Various delivery systems are known and can be used to administerpeptide-based (such as AAT-1) and non-peptide active agents astherapeutics. Such systems include, for example, encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing therapeutic molecule(s) (see, e.g., Wu et al., J. Biol. Chem.262, 4429, 1987), construction of a therapeutic nucleic acid as part ofa retroviral or other vector, and the like. Although current AAT-1formulations are administered to subject intravenously, variousalternative methods of administration of AAT-1 or additional activeagents include, but are not limited to, intrathecal, intradermal,intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous,intranasal, epidural, and oral routes. The active agent therapeutics maybe administered by any convenient route, including, for example,infusion or bolus injection, topical, absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,and the like) ophthalmic, nasal, and transdermal, and may beadministered together with other biologically active agents. Pulmonaryadministration can also be employed (e.g., by an inhaler or nebulizer),for instance using a formulation containing an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thedescribed compositions by injection, catheter, suppository, or implant(e.g., implants formed from porous, non-porous, or gelatinous materials,including membranes, such as sialastic membranes or fibers), and thelike. In another embodiment, therapeutic agents are delivered in avesicle, in particular liposomes (see, e.g., Langer, Science 249, 1527,1990; Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365,1989).

In yet another embodiment, any one of the agents described herein can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see, e.g., Langer Science 249, 1527, 1990; Sefton Crit. Rev.Biomed. Eng. 14, 201, 1987; Buchwald et al., Surgery 88, 507, 1980;Saudek et al., N. Engl. J. Med. 321, 574, 1989). In another embodiment,polymeric materials can be used (see, e.g., Ranger et al., Macromol.Sci. Rev. Macromol. Chem. 23, 61, 1983; Levy et al., Science 228, 190,1985; During et al., Ann. Neurol. 25, 351, 1989; Howard et al., J.Neurosurg. 71, 105, 1989). Other controlled release systems, such asthose discussed in the review by Langer (Science 249, 1527 1990), canalso be used.

As described above, in particular examples wherein AAT-1 is administeredwith at least one additional active agent, the active agents areadministered simultaneously, and by the same mode of administration. Inother examples, the pharmaceutical compounds are administered atdifferent times, and either by the same or different more ofadministration.

The vehicle in which the agent is delivered can include pharmaceuticallyacceptable compositions of the compounds, using methods well known tothose with skill in the art. For instance, in some embodiments,described active agents typically are contained in a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable, or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil, and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions, blood plasma medium, aqueous dextrose,and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. The medium may also containconventional pharmaceutical adjunct materials such as, for example,pharmaceutically acceptable salts to adjust the osmotic pressure, lipidcarriers such as cyclodextrins, proteins such as serum albumin,hydrophilic agents such as methyl cellulose, detergents, buffers,preservatives and the like.

Examples of pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The therapeutic, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. The therapeutics can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations, and the like. The therapeutic can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. A morecomplete explanation of parenteral pharmaceutical carriers can be foundin Remington: The Science and Practice of Pharmacy (19th Edition, 1995)in chapter 95.

Embodiments of other pharmaceutical compositions are prepared withconventional pharmaceutically acceptable counter-ions, as would be knownto those of skill in the art.

Therapeutic preparations will contain a therapeutically effective amountof at least one active ingredient, preferably in purified form, togetherwith a suitable amount of carrier so as to provide proper administrationto the patient. The formulation should suit the mode of administration.

The compositions of this disclosure can be formulated in accordance withroutine procedures as pharmaceutical compositions adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the compositions may also include asolubilizing agent and a local anesthetic such as lidocaine to ease painat the site of the injection.

The ingredients in various embodiments are supplied either separately ormixed together in unit dosage form, for example, in solid, semi-solidand liquid dosage forms such as tablets, pills, powders, liquidsolutions, or suspensions, or as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where one or more ofthe indicated agents is to be administered by infusion, it can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where one or more of the indicated agents is tobe administered by injection, an ampoule of sterile water or saline canbe provided so that the ingredients may be mixed prior toadministration.

Effective amounts can be determined by standard clinical techniques. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and should be decided according to the judgmentof the health care practitioner and each patient's circumstances.Exemplary dosages of the individual compounds are described herein, butmyriad other dosage regimens are encompassed by this disclosure. Anexample of an additional dosage range is 0.1 to 200 mg/kg body weight insingle or divided doses. Another example of a dosage range is 1.0 to 100mg/kg body weight in single or divided doses.

In particular embodiments. AAT-1 is provided in a composition at aconcentration of 1 gram in 50 cc sterile fluid in the form of a sterileor physiologically isotonic aqueous solution. In particular embodimentsa single dosage of AAT-1 administered to a subject at a dosage of 30 mgto 400 mg per kg body weight per day, such as 60 mg to 100 mg per kgbody weight per day. In other embodiments, multiple comparable dosagesof AAT-1 are administered to a subject in a combination of dosingperiods prior to, during, or after cardiac surgery.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, and severity of the condition of the host undergoingtherapy.

In some embodiments, sustained localized release of the pharmaceuticalpreparation that comprises a therapeutically effective amount of atherapeutic compound or composition may be beneficial. Slow-releaseformulations are known to those of ordinary skill in the art. By way ofexample, polymers such as bis(p-carboxyphenoxy)propane-sebacic-acid orlecithin suspensions may be used to provide sustained localized release.

It is specifically contemplated in some embodiments that delivery is viaan injected and/or implanted drug depot, for instance comprisingmulti-vesicular liposomes such as in DepoFoam (SkyePharma, Inc, SanDiego, Calif.) (see, for instance, Chamberlain et al., Arch. Neuro.50:261-264, 1993; Katri et al., J. Pharm. Sci. 87:1341-1346, 1998; Ye etal., J. Control Release 64:155-166, 2000; and Howell, Cancer J.7:219-227, 2001).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Single Dose Administration of Alpha-1 Anti-Trypsinfor Treatment of Organ Injury and Postoperative Bleeding in PatientsUndergoing Cardiac Surgery with Cardiopulmonary Bypass

This example describes assay of AAT-1 as an effective inhibitor ofinjury to a subject undergoing cardiac surgery involving cardiopulmonarybypass. In particular, methods for determining the effect of AAT-1 onpostoperative blood loss and organ-function assessment are described.

Methods AAT-1 Dosage

Previous studies and clinical practice indicated that the administrationof multiple intravenous AAT-1 doses of 60 mg per kg body weight is safe(Wewers M D, et al., N Engl J Med., 316:1055-62, 1987). Such doing wasfound result in a low incidence of side-effects, with those reportedbeing benign in nature. Based on pharmacokinetic studies, intraoperativeadministration of AAT-1 dosage (60 mg/Kg) results in AAT-1 plasma levelswhich resemble acute phase response; immediately followingadministration (Wewers M D, et al., N Engl J Med., 316:1055-62, 1987). A30% reduction in plasma levels is anticipated after termination of CPBwith gradual return to normal preoperative AAT-1 levels afterwards(Pickering N J et al., Am J Clin Pathol, 80:459-64, 1983).

Determination of Study Eligibility

Patients eligible in a clinical study to assay use of AAT-1 are male orfemale, 40-70 years of age. Eligible patents arc candidates for isolatedcoronary artery bypass grafting (CABG) employing cardiopulmonary bypass(CPB), have a calculated logistic Euroscore risk stratification of 5% orless, and will provide signed patient's written informed consent.

For the initial study, exclusion criteria will be based on presence ofco-existing conditions including: coagulation abnormalities, severepulmonary disease defined by blood oxygen saturation of 90% or less orFEV1 of less than 60% of predicted, renal dysfunction defined be serumcreatinine levels higher or equal to 1.8 mg %, abnormal liver functiontests, uncontrolled diabetes mellitus, severe peripheral vasculardisease, a prior cerebrovascular neurological event, abnormal left orright ventricular function, and/or treatment with warfarin orthienopyridine class of anti-platelet agents.

The study participants are randomized to receive either single doseAAT-1 60 mg per kg or placebo.

Trial Medication Administration

Preparation and dosing of AAT-1 are performed by an unblindedpharmacist. The medication is diluted just prior to administration, andis selected from a commercially available AAT-1 preparation. Thepatients, research staff, laboratory personnel and data analysts remainblinded to the identity of the treatment from the time of randomizationuntil database lock. Data unblinding is unnecessary. A randomizationlist is produced by the pharmacist, and was secured and confidentialuntil time of unblinding.

The placebo solution comprises human albumin that resembles the colorand consistency of the AAT-1 solution.

The medication is given 3-5 hours prior to surgery (skin incision).Administration rate of the drug does exceed 0.04 ml per kg per minute(for approximately 60-80 minutes). Vital signs including blood pressure,pulse rate and body temperature are correspondingly monitored.

Surgical Technique

Consistent with study center policy, fentanyl citrate (20-50 mcg/kg),midazolam (2-3 mg) and isoflurane (0.5-2%) are used for induction andmaintenance of anesthesia.

Standard median sternotomy is performed followed by harvesting of bypassconduits, uni- or bilateral internal thoracic artery, radial artery orsaphenous vein graft. Heparin loading dose is administered prior toinitiation of cardiopulmonary bypass (CPB) to achieve kaolin activatedcoagulation time (ACT). Standard ascending aorta—right atrialcannulation is performed to institute CPB. CPB is initiated afterverifying ACT level of 480 seconds or more and periodically monitored.Standard centrifugal pump and a membrane oxygenator are used forextracorporeal circulation (CPB). Consistent with standard technique,active systemic cooling is avoided and patients' core temperature rangesbetween 32 and 37° C. Distal anastomoses are performed during singleaortic cross-clamp and blood cardioplegic arrest. Proximal anastomosesare performed during single aortic cross-clamp. Cold (10° C.) bloodcardioplegic solution is delivered in a 4:1 ratio, in antegrade fashionvia the aortic root with or without additional retrograde administrationvia the coronary sinus. After cardioplegic induction (10 ml / Kg)intermittent doses (300-500 ml) are administered; following completionof each distal anastomosis. Heparin is reversed using protamine sulphatein a ratio of 1:1 after weaning from CPB.

Data Collection

Preoperative data: Demographic, morphological and clinical descriptorsincluding age, gender, body mass index (BMI), body surface area (BSA)co-morbidity, Euroscore risk-stratification, medication, etc. isrecorded. Preoperative laboratory analysis includes complete bloodcount, coagulation profile, serum creatinine levels and creatinineclearance, liver function test and arterial blood gases test andserology for HIV, HCV, HBC. Compatible with our routine policy, allpatients underwent preoperative echocardiography, coronary angiography,chest x-ray, lung function tests (spirometry) and carotid artery duplexstudy. Study participants are assigned to undergo preoperative brainMRI; and are subjected to the protocol described below.

Intraoperative: The type of surgery is categorized. The following datais recorded: heparin dose given prior to bypass initiation; activatedclotting time (ACT) counts during the operation (prior, during and afterCPB); operative time, cross-clamp time and CPB time; number of trials towean from CPB, type of inotropes and dosage used during weaning fromCPB; blood products utilization during surgery. Allergic reactions oradverse events observed by the surgeon or anesthesiologist aredocumented. The individual surgeon's impression regarding bleedingtendency is recorded.

Postoperative Organ Function and Blood Loss Evaluation

The occurrence and magnitude of systemic inflammatory response and organdysfunction are recorded and quantified by laboratory markers. Relatedlaboratory markers arc monitored on a daily basis during the recoveryperiod (in the intensive care unit and at the ward). The followingorgans and corresponding markers are monitored: Pulmonary function:Pulmonary function is evaluated by measured overall mechanicalventilation time, peak inspiratory pressures (PIP), plateau pressures,physiologic dead space and static and dynamic lung compliance.Bronchoalveolar lavage (BAL) is performed 3 hours after operation (whilethe patient is anesthesized and intubated) and extracted fluid isanalyzed for inflammatory markers. A-a DO2 calculation[AaDO₂=(713×FiO₂)−(pCO₂/0.8)−(paO₂)] is measured daily. Completepulmonary function test is performed before and 4 days after theoperation. Chest radiographs are evaluated and quantified by anindependent radiologist for the occurrence of atelectasis, pulmonaryedema, or pleural changes.

Renal function: Renal function is evaluated by daily measurements ofurine output, serum creatinine levels, creatinine clearance and urinaryalbumin levels. Acute kidney injury (AKI) markers are sampled in theICU.

Brain injury assessment: The degree of insult to the brain is measuredby plasma S-100 protein levels. Assessment of damage to the blood-brainbarrier (BBB) is performed by magnetic resonance imaging (MRI) modalityon post-operative days 1 and day 5. (see technique protocol below).

Hepatic function: Determined by daily measurements of serum hepaticenzymes levels.

Cardiac function: Cardiac function is monitored by assaying cardiacenzyme levels; need and magnitude of required inotrope treatment;occurrence of low cardiac output syndrome (defined as systolic bloodpressure of 90 mmHg or less coupled with central venous pressure (CVP)of 15 mmHg or more), and incidence of cardiac arrhythmias. Transthoracicechocardiography examination is performed on postoperative day 5 andassessed by an independent cardiologist.

Blood loss: Operative and postoperative blood loss is monitored as wellas daily hemoglobin levels. Daily platelet counts and thromboelastogramsare performed. The distribution of blood products and total administeredare recorded daily. Postoperative CRP levels are evaluated daily.

Blood sampling and laboratory analysis methods for cytokine levels: 10mL whole blood venous EDTA samples are collected from radial arterycatheter at five specified occasions: before induction of anaesthesia,30 minutes after aortic cross-clamp positioning, and 3, 6, and 9 hoursafter aortic cross clamp positioning. The blood samples are subsequentlycentrifuged at 4° C. for 15 min and the serum stored at −70° C. Samplesare analyzed for the following markers: Polymorphonuclear NeutrophilElastase (PMNE), Interleukin-1α (IL-1α), Interleukin-1β (IL-1β),Interleukin-2 (IL-2). Interleukin-4 (IL-4), Interleukin-6 (IL-6),Interleukin-8 (IL-8), Interleukin-10 (IL-10). Interferon-γ (IFN-γ),Tumor Necrosis Factor-α (TNF-α), Vascular Endothelial Growth Factor(VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), and EndothelialGrowth Factor (EGF).

Daily blood samples are collected postoperatively for platelet count,renal function, liver function, CRP levels S-100 protein and troponin.Post-operative day 1 urine samples for acute kidney injury markers(N-GAL, KIM, Cistatin C) are collected.

Early post-operative adverse events are documented. These include 30-daymortality, new neurological events, myocardial infarction, renaldysfunction, need for re-exploration for bleeding and deep sternal woundinfection.

Blood-Brain Barrier (BBB) assessment by MRI: The imaging modality usedfor BBB assessment is through use of a MRI scanner (Philips 3T orGeneral Electric 1.5T). The examination format includes 24 cm FOV, 35contiguous interleaved slices, 3.5-4 mm thick and co-localized acrossseries. Trace-weighted DWI images are obtained at b=1000 from a 13-15direction DTI sequence with an in-plane resolution of 2.5×2.5 mm andTR/TE=10 s/58 ms at 3T or TR/TE=10 s/72 ms at 1.5T. T2-FLAIR images areobtained with an in-plane resolution of 0.94×0.94 mm, TR/TE=9000/120 msand TI=2600 ms at 3T or TR/TE=9000/140 ms and TI=2200 ms at 1.5T. (25).

Results

10 patients are recruited to the study. Five patients receive AAT-1prior to surgery, and five patients receive placebo. Both groups arecomparable with regard to preoperative and operative descriptors(parameters: age, sex, past medical history, left ventricular function,number of grafts performed during surgery and cardiopulmonary bypasstime). There are no major operative events and postoperativecomplications are not observed in the cohort patients. Physicalmeasurements described below are intended as approximations of expectedresults.

Inflammatory Parameters

Intra- and postoperative blood levels of IL-6, TNF-alpha, IL-1 beta,IL-8, MCP-1, LDH, and D dimmer increase in both groups. Elevation ofthese cytokines is significantly higher in the placebo group.

Nervous System

MRI at postoperative day (POD) 1 and POD 5 show significant BBBdisruption (MR imaging-detected) in 60% of patients in the placebocompared to only 20% in the AAT-1 group. Acute major neurologicaldeficit events are not detected in any patient.

S-100 protein on POD 1 increases by 3-fold in the placebo group comparedto 1.5-fold in the AAT-1 group.

Respiratory System:

Postoperative BAL show significant increase in neutrophil elastase andTNF-alpha counts. Increase of these cytokines is twice more prominent inthe placebo group.

Postoperative IL-8 levels decrease more in the placebo group. A-a DO2decreases in all patients after surgery, more in the placebo group. TheA-aDO2 returns to preoperative values on POD 3 in the AAT-1 groupcompared to POD 5 in the placebo group.

Lung function tests show substantial decrease in FEV-1 and TLC on POD 4in the placebo group compared to almost no decrease in the AAT-1 group.

Postoperative chest x-rays show atelectasis in 3 of the 5 placebo groupand non in the AAT-1 group.

Cardiovascular System

No signs of low cardiac output syndrome are recorded in any of thepatients. Postoperative echocardiography show normal cardiac function inall patients.

Urinary System

Average preoperative creatinine is 1.0 mg/dL in both groups of patients.Postoperatively, the average creatinine rises to 1.3 mg/dL in theplacebo group and remains 1 mg/dL in the AAT-1 group. Acute kidneyinjury markers, N-GAL KIM and Cystatin C, increase after surgery in allpatients. The increase is substantially higher in the placebo group.

Fluid retention after operation as measured by daily body weight is moreprominent in the placebo group (postoperative maximal increase in bodyweight was twice as much in the placebo group).

Liver Function

Blood liver enzymes levels increase postoperatively only in the placebogroup

Post Operative Bleeding and Thrombocytes Function

Operative ACT levels are similar in both groups.

Post-operative bleeding is substantially lower in the AAT-1 group both 6hours and 24 hours post operatively. Blood D dimmer levels as a markerof fibrinolysis increases more in the placebo group.

Postoperative thromboelastography shows signs of thrombocytesdysfunction in all patients (prolonged K and decreased MA); and more inthe placebo group.

The AAT-1 group receives post-operatively about half the amount of bloodproducts as compared to the placebo group.

Operative bleeding is also assessed by the individual surgeons'impression blinded to the medication and scaled from 1 to 10. Theresults show that patients in the AAT-1 group tended to bleed less.

Conclusion

The results of this randomized placebo controlled pilot study indicatethat AAT-1 (administered and dosed as described) substantially attenuateCPB-inflicted organ injury. Post-operative bleeding and correspondingneed for post-operative blood product administration are reduced.Administration of AAT-1 also appears to reduce post-CPB inflammation.Hospital length of stay is reduced reflecting improved overall patients'outcome.

Example 2 Multiple Dose Administration of Alpha-1 Anti-Trypsin (AAT-1)for Treatment of Organ Injury and Post-Operative Bleeding in PatientsUndergoing Cardiac Surgery with Cardiopulmonary Bypass

This example describes treatment of post-operative bleeding and organdamage resultant from cardiac surgery by administrations of multipledoses of AAT-1.

Except as specified herein, all methods are as described in Example 1.

As described above, AAT-1 is used to treat or prevent injury resultantfrom cardiac surgery with cardiopulmonary bypass. Example 1 describessuch treatment with a single dose of a composition comprising AAT-1. Inthe current example, patients are administered two equivalent doses ofAAT-1. As described in Example 1, the first dose is administered to thepatient as part of the preoperative procedure. Following surgery, thesubject is monitored for excessive bleeding and organ injury asdescribed. At post-operative day 1-4, subjects presenting symptomsindicative of excessive bleeding and organ injury are administered asecond dose of the composition comprising AAT-1.

Example 3 Combination Treatment of Organ Injury and Post-OperativeBleeding in Patients Undergoing Cardiac Surgery with CardiopulmonaryBypass

In this example, damage to a subject resultant from use ofcardiopulmonary bypass in cardiac surgery is treated by administering toa subject a combination of AAT-1 and aminocaproic acid.

Methods are as described in the previous examples. In the currentexample, a subject undergoing cardiopulmonary bypass is administered acomposition comprising AAT-1 as part of preoperative treatment.Following surgery, the subject is monitored as described for excessivebleeding and organ damage. A subject presenting symptoms of damage tothe respiratory system, urinary system or nervous system is administereda second dose of AAT-1 at 60 mg per kg body weight in a compositioncontaining an effective amount of aminocaproic acid for additional,complimentary treatment.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. I thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1.-11. (canceled)
 12. A method for treating or preventing injury duringor resulting from cardiac surgery in a subject, the method comprisingadministering to the subject in need thereof a composition comprising atherapeutically effective amount of alpha-1 antitrypsin (AAT-1), orfunctional variant thereof.
 13. The method of claim 12, wherein theinjury results from use of cardiac bypass.
 14. The method of claim 12,wherein the organ injury comprises bleeding.
 15. The method of claim 12,wherein the composition is administered to the subject before thecardiac surgery, during the cardiac surgery, after the cardiac surgeryor a combination thereof.
 16. The method of claim 12, wherein theconcentration of AAT-1 in the composition is 1 gram in 50 cc sterilefluid.
 17. The method of claim 12, wherein the AAT-1 is administered tothe subject at a concentration of 30 to 300 mg per kg body weight perday.
 18. The method of claim 12, wherein the AAT-1 is administered tothe subject at a concentration of 60 to 100 mg per kg body weight perday.
 19. The method of claim 12, wherein the AAT-1 is administered in asingle dose.
 20. The method of claim 12, further comprising at least oneadditional active ingredient.
 21. The method of claim 20, wherein the atleast one additional ingredient is administered to the subject prior to,concurrent with, or after administration of the composition comprisingAAT-1.
 22. The method of claim 12, wherein the subject is a human or aveterinary subject.
 23. The method of claim 20, wherein the at least oneadditional active ingredient is for the prevention, treatment, or bothof excessive post-operative bleeding and/or organ damage.
 24. The methodof claim 20, wherein the at least one additional active ingredient is:plasma, platelets, cryoprecipitant, alpha aminocaproic acid, or anycombination thereof.
 25. The method of claim 12, wherein the compositionis an injectable composition.
 26. The method of claim 12, wherein thecardiac surgery is selected from the group comprising: coronary arterybypass grafting surgery, aortic valve replacement or repair, mitralvalve replacement or repair, tricuspid valve replacement or repair,ascending aorta replacement, heart transplantation, lung transplantationor any combination thereof.